Isolation of cyanide-degrading bacteria andmolecular characterization of its
cyanide-degrading nitrilase

Ayane Terada1
Daisuke Komatsu1,2Takahiro Ogawa1
Darin Flamandita3
Muhamad Sahlan 3
Minoru Nishimura2
Masafumi Yohda, Dr.
∗
Professor 1

1 Department of Biotechnology and Life Science, Tokyo University ofAgriculture and Technology, Koganei, Tokyo, Japan
2 EnBio Engineering, Chiyoda, Tokyo, Japan
3 Department of Chemical Engineering, Universitas Indonesia, Depok,

Indonesia

Abstract
Hydrogen cyanide is an industrially important chemical, andits annual production is more than 1.5 million tons. Because of
its toxicity, the cyanide-containing effluents from industries
have caused many environmental problems. Among various
methods to treat the contaminated soils or water, thebiological degradation is regarded to be promising. We
isolated two cyanide-degrading microorganisms, Pedobacter
sp. EBE-1 and Bacillus sp. EBE-2, from soil contaminated withcyanide. Among these bacteria, Bacillus sp. EBE-2 exhibited
significantly a high cyanide-degrading ability. Bacillus sp.
EBE-2 might be used for the remediation of cyanide
contaminated water or soil. A nitrilase gene was cloned from

Bacillus sp. EBE-2. Bacillus nitrilase was expressed in
Escherichia coli and purified. Bacillus nitrilase exhibitedcyanide-degrading activity as a large oligomer. Since formic
acid formation from cyanide was observed, Bacillus nitrilase islikely to be a cyanide hydrolase. Although there exist various
homologous enzymes annotated as carbon–nitrogen familyhydrolases, this is the first report on the cyanide degrading
activity. The structure and catalytic site of Bacillus nitrilase
were studied by homology modeling and molecular docking
simulation. © 2020 International Union of Biochemistry and Molecular
Biology, Inc. Volume 00, Number 0, Pages 1–7, 2021

Keywords: cyanide, Bacillus, nitrilase, biocatalysis, biodegradation, protein
modeling

1. Introduction
Cyanide is a compound containing the C≡N group. The cyanide
anion is highly toxic. It binds to iron at the catalytic center ofthe mitochondrial protein cytochrome c oxidase and prevents
electron transport from cytochrome c to oxygen [1]. As a result,

Abbreviations: CNO, cyanide oxygenase; CynD, cyanidase..
∗ Address for correspondence: Masafumi Yohda, Department of
Biotechnology and Life Science, Tokyo University of Agriculture andTechnology, 2-24-16 Naka-cho, Koganei, Tokyo 184–8588, Japan. Tel.: +81
42 388 7479; Fax: +81 42 388 7479; e-mail: yohda@cc.tuat.ac.jp
Present address of Daisuke Komatsu: Shimizu Cooperation, 2-16-1
Kyobashi, Chuo-ku, Tokyo 104–8370, Japan.
Ayane Terada and Daisuke Komatsu contributed equally to this work.
Received 13 October 2020; accepted 25 December 2020
DOI: 10.1002/bab.2095
Published online in Wiley Online Library(wileyonlinelibrary.com)

the electron transport chain is disrupted. Thus, cells can nolonger aerobically produce ATP for energy. Hydrogen cyanide(HCN) is an industrially important chemical and has been usedin the pharmaceutical industry, polymer manufacturing, mining and steel manufacturing, electroplating, and agrochemical
production. More than 1.5 million tons of HCN are produced
annually. The cyanide-containing effluents from such industries have caused many environmental problems. Therefore,various cyanide detoxification strategies have been developed.
Chemical and physical treatments are effective in the detoxification process of cyanide [2]. However, the cyanide-degrading
abilities affected by multiple factors, such as the chemicalcomposition of the waste, the amount of effluent, and the areaof contamination. Microbial degradation of cyanide from ore
mining wastewater is regarded as the most successful method
for cyanide removal [3, 4].
In the biodegradation process, free cyanide and metal
cyanide complexes are transformed into ammonia and1

Biotechnology andApplied Biochemistrybicarbonate. Because various cyanide compounds are produced in nature, multiple microorganisms, including fungi,
algae, yeasts, and bacteria, can degrade cyanide compounds.
Many fungal species, including the genera Fusarium, Penicillium, Scytalidium, and Trichoderma, are known to degrade
cyanide [5, 6]. The yeast Cryptococcus cyanovorans could
utilize up to 10 mM NaCN as a sole carbon source [7]. Bacterial
species belonging to the genera Pseudomonas, Arthrobacter,
Acinetobacter, Alcaligenes, Bacillus, Rhodococcus, and Serratia
have been reported to exhibit cyanide-degrading ability [8–12].
Varieties of enzymatic pathways are involved in theprocesses for the biotransformation and biodegradation of
cyanide compounds. Cyanide compounds are degraded byfour different pathways: the oxidative pathway, hydrolytic
pathway, reductive pathway, and substitution pathway [13]. In
the aerobic pathway, cyanide is converted into ammonia andcarbon dioxide using three different enzymes, namely, cyanase,cyanide monooxygenase, and cyanide dioxygenase [3, 14].
Among these enzymes, cyanide monooxygenase and cyanide
dioxygenase can degrade free cyanide. Cyanase catalyzes thedegradation of cyanate, which is produced from cyanide byoxidation in soil. Using bicarbonate as a nucleophile reactant,cyanase converts cyanate to an unstable carbamate, whichfurther spontaneously breaks down to form ammonia andcarbon dioxide by decarboxylation [15]. Cyanide oxygenase(CNO) from Pseudomonas fluorescens NCIMB 11764 is apterin-dependent enzyme that catalyzes the mono-oxygenase
conversion of cyanide to formic acid and ammonia [16].
CNO consists of four protein components (P1–P4). P1 is amultimeric, 230-kDa flavoprotein exhibiting the properties ofa peroxide-forming NADH oxidase (oxidoreductase). P2 is a
NADH peroxidase. P3 is a sizable oligomeric protein (∼300 kDa)
with cyanide dihydratase activity. The remaining P4 component
is a carbonic anhydrase [17]. Cyanide dioxygenase is also
thought to be a pterin-dependent enzyme [14]. Most microbes
exploit the hydrolytic pathway for the degradation of cyanide
compounds. Various hydrolases and nitrilases can degrade
free as well as organic cyanide compounds. In the hydrolytic
pathway, direct cleavage of the carbon–nitrogen triple bondby nitrilase family enzymes results in the formation of thecorresponding amide or acid and ammonia [18]. Nitrilases
are generally homo-oligomers of an αββα–αββα sandwich
fold protein and use the triad of cysteine, glutamic acid, andlysine as the active center [19]. Among nitrilases, cyanidase(cyanide dehydratase, CynD) and cyanide hydratase candegrade free cyanide. Cyanide hydratase exists throughout
the fungal kingdom and converts cyanide into formamide as
the product [20, 21]. In the cell, cellular amidases normallyconvert formamide into formate and ammonia. CynD exhibitshigh sequence homology with other bacterial nitrilases withdifferent substrates [22]. Cyanide hydratase is also homologous
to nitrilases. However, the sequence identity between CynD andcyanide hydratase is only approximately 25%–30%. CynD andcyanide hydratase are more similar to other nitrilases than toeach other.

2

Nitrogenase works in the reductive pathway. Nitrogenase reduces cyanide into methane and ammonia [13].
In substitution pathway, cyanides are converted to thiocyanate by the transfer of a sulfur group from thiosulfate or
3-mercaptopyruvate by rhodanese or 3-mercaptopyruvate sulfurtransferase. The produced thiocyanate is further assimilated
as a nitrogen source [13].
In this study, we isolated two cyanide-degrading microorganisms, Pedobacter sp. and Bacillus sp., from soil contaminated with cyanide. Among these species, Bacillus sp. exhibited
significantly high cyanide-degrading ability. Bacillus sp. has thenitrilase gene. Bacillus nitrilase was expressed and purified.
It was found to be a cyanide hydrolase. The structure andcatalytic site were studied by homology modeling.

2. Materials and Methods
2.1. Isolation of cyanide-degrading bacteria
Groundwater obtained from a site contaminated with cyanide
was used as the source of cyanide-degrading bacteria. The
groundwater was supplemented with free cyanide in the form
KCN at approximately 12 mg/L. The sample was incubatedwith or without the addition of 0.1% yeast extract. The numberof bacteria was counted as colony forming units. The cyanide
concentration was measured by the chloramine T/isonicotinicacid/1,3-dimethylbarbituric acid method using visocolor ECO
cyanid (MACHEREY-NAGEL, Düren, Germany) with slight
modification. The cyanide-degrading bacteria were isolated byseveral repeated of plating and cyanide degradation activity
assays.

2.2. 16S rRNA gene cloning and sequence
The 16S rRNA gene was amplified by PCR using the primers
16SrRNA_Fw5 -AGAGTTTGATCCTGCCTCAG-3 and 16SrRNA_Rv 5 -GGTTACCTTGTTACGACTT-3 . The amplified DNAs
were cloned into the pMD20 vector by TA cloning and appliedfor DNA sequencing.

2.3. Cloning, expression, and purification of Bacillus
nitrilase
The Bacillus nitrilase gene was obtained by PCR using theprimers BNit_Fw 5 -CATATGAAAATCGCCCTTGT-3 and BNit
_Rv5 -CTCGAGCTAATAGATATCCAGACGCTGA-3 and cloned
into the pMD20 vector. After sequence analysis, the gene wascloned into the NdeI/XhoI site of the pET23b vector (pET-BNit).
E. coli BL21 (DE3) transformed with pET-BNit was cultured in
LB media supplemented with 100 μg/mL ampicillin at 18 °C for
70 H. The cells were harvested by centrifugation at 5,000g for
10 Min at 4 °C. The harvested cells were suspended in buffer(50 mM Tris–HCl, pH 8.0) and disrupted by sonication, andthe suspension of disrupted cells was centrifuged at 24,000g
for 30 Min at 4 °C. The supernatant was applied to an anionexchange column (TOYOPEARL DEAE-650M; Toyobo, Osaka,
Japan) equilibrated with buffer A (50 mM Tris–HCl, pH 8.0,
1 mM EDTA, and 1 mM DTT). Proteins were eluted with a linear
gradient of NaCl in buffer A. Fractions containing Bacillus

Cyanide degrading bacteria and nitrilase

(b)(mg/l)(cfu/mL)

(a)

(day)

FIG. 1

Enhancement of cyanide degradation with thegrowth of bacteria by the addition of yeast extract.
The groundwater was supplemented with freecyanide as KCN to be approximately 12 mg/L. The
water was incubated with or without the additionof 0.1% yeast extract. Growth of bacteria (a) anddecrease of cyanide (b) are shown.

(mg/L)(b)(mg/L)(a)(day)

FIG. 2(day)

Cyanide degradation abilities of isolated bacteria.
The cyanide-degrading activities of the isolated
bacterial strains were investigated by incubatingthem in physiological salt solutions containing
KCN. (a) Pedobacter sp. EBE-1. The initial celldensity was 3 × 109 CFU/mL. (b) Bacillus sp. EBE-2.
The initial cell density was 2 × 106 CFU/mL.

nitrilase were pooled and dialyzed with buffer A overnight.
The dialyzed protein solution was applied to a RESOURCE
Q column (Cytiva, Tokyo, Japan) equilibrated with buffer A.
Proteins were eluted with a linear gradient of NaCl in buffer
A. Fractions containing Bacillus nitrilase were concentrated byultrafiltration (Amicon Ultra, Merck Millipore, Billerica, CA) andthen applied to a HiLoad 26/60 Superdex 200 pg size-exclusion
column (Cytiva) equilibrated with buffer B (50 mM Tris–HCl, pH
7.5, 0.1 mM EDTA, and 150 mM NaCl).

Biotechnology and Applied Biochemistry(H)

2.4. Cyanide degradation assay
Cyanide-degrading activity was assayed by mixing the Bacillus
nitrilase solution with 270 ng of KCN in 1.5 mL of buffer A.
The decrease in cyanide was measured after incubation at
30 °C for 30 Min. The cyanide hydrolase activity was measured
as follows. The reaction was started with 100 μL of Bacillus
nitrilase (0.8 mg/mL in buffer A) and 1.4 mL of reaction
solution [676 ng of KCN, 6.9 μM NAD+ , and 1 unit of formic aciddehydrogenase (Sigma–Aldrich Japan, Tokyo, Japan) in buffer
A]. Formation of formic acid was measured by the increase in
NADH absorbance at 340 nm.

2.5. Homology modeling
The three-dimensional structure of Bacillus nitrilase wasconstructed by the automated protein modeling server SWISSMODEL [23] on the basis of homology modeling with reference3

Biotechnology andApplied Biochemistry

kDa

(b)

E. coli/ E. coli/
Nitrilase Control
S P S P

45
Nitrilase

31(mg/L)

(a)

(H)

(d)

(c)

(mL)

FIG. 3

Expression, cyanide-degrading activity, andpurification of Bacillus nitrilase. (a) SDS-PAGE
analysis of the expression of Bacillus nitrilase. S,
supernatant of crude extract; P, precipitate of crude
extract. (b) Cyanide-degrading activity ofrecombinant E. coli expressing Bacillus nitrilase.
(c) Size-exclusion chromatography of the purified
Bacillus nitrilase. (d) Cyanide hydrolase activity ofBacillus nitrilase. Formation of formic acid fromcyanide was observed by the production of NADH.

to the crystal structure of the putative carbon–nitrogen familyhydrolase from Staphylococcus aureus (PDB ID: 3P8K), whichshared the highest sequence identity to that of Bacillus nitrilase.

2.6. Molecular docking
Molecular docking was performed using AutoDock Vina [24]with preprocessing of structures using AutoDock Tools. Cyanide
ion was docked to the modeled structure of Bacillus nitrilase
within a box (with dimensions of 40 Å × 40 Å × 40 Å).

3. Results and Discussion
3.1. Isolation of cyanide-degrading bacteria
To observe microbial degradation of cyanide in the groundwater obtained from the site contaminated with cyanide, theeffect of the addition of yeast extract was investigated. The

4

(Min)

groundwater was supplemented with free cyanide as KCN tobe approximately 12 mg/L. The water was incubated with orwithout the addition of 0.1% yeast extract. An increase in bacteria occurred when the groundwater was supplemented withyeast extract (Fig. 1a). As bacteria grew, cyanide concentration
decreased in the condition with yeast extract (Fig. 1b). On thecontrary, no bacterial growth and a decrease of cyanide wereobserved without yeast extract (Figs. 1A and 1b). We thentried to isolate cyanide-degrading bacteria from the culture
by repeated plating and cyanide degradation activity assays.
Finally, we isolated two different cyanide-degrading bacteria.
The 16S rRNA sequences showed that one strain belongs
to the genus Pedobacter, and the other strain is Bacillus
sp. Pedobacter sp., named Pedobacter sp. EBE-1, was highly
homologous to P. heparinus strain DSM 2366 [25]. The Bacillus
sp., named Bacillus sp. EBE-2, was similar to B. pumilus strain
PDSLzg-1 [26].
The cyanide-degrading activities of these bacterial strains
were investigated by incubating Pedobacter sp. EBE-1 (Fig. 2a)and Bacillus sp. EBE-2 (Fig. 2b) in physiological salt solutions
containing KCN. Among them, Bacillus sp. EBE-2 showed
relatively higher activity than that of the other strain. The
Bacillus sp. EBE-2 could degrade 80 mg CN− /L in 2 H by
2 × 106 CFU/mL. Previously, 13 bacterial species were isolated
from electroplating wastewater to assess their ability tobiodegrade free cyanide [27]. A mixed culture mainly dominatedCyanide degrading bacteria and nitrilase

FIG. 4

Sequence alignment of Bacillus nitrilase with other
nitrilases. Amino acid sequence of Bacillus
nitrilase (Bacillus_Nitrilase) was aligned with other
nitrilase family proteins (A0A0H2WXM0,
Staphylococcus aureus hydrolase,carbon–nitrogen family; Q9UYV8, Pyrococcus
abyssi nitrilase; Q5JEU9, Thermococcus
kodakarensis carbon–nitrogen hydrolase; B1L5H4,
Korarchaeum cryptofilum nitrilase/cyanide
hydratase; Q2LUZ0, Syntrophus aciditrophicus
carbon–nitrogen hydrolase family protein;
Q2S2E4, Salinibacter ruber hydrolase,carbon–nitrogen family). The putative catalytic
regions are boxed. The residues constituting thecatalytic triad are marked with #.

by Bacillus sp. (B. safensis, B. lichenformis, and B. tequilensis)
could degrade 131 (65.5%) and 177 (44.3%) mg CN− /L in
cultures containing 200 and 400 mg CN− /L over a period of 8days, respectively. Therefore, Bacillus sp. EBE-2 seems to havesignificantly high cyanide-degrading activity.

3.2. Cloning, sequencing, expression, and purificationof Bacillus nitrilase
Then, we tried to find the enzyme responsible for cyanide
degradation from Bacillus sp. EBE-2. Although some B.

Biotechnology and Applied Biochemistry

pumilus strains are known to have the CynD gene and exhibit cyanide-degrading activity, the CynD gene does not exist
in the genome of the B. pumilus strain PDSLzg-1. A nitrilase
gene (WP_047202096.1) was observed in the genome of B.
pumilus strain PDSLzg-1. Considering that nitrilase is responsible for cyanide degradation, we tried to clone the nitrilase
gene from Bacillus sp. EBE-2. Expecting high sequence identity with the nitrilase from B. pumilus strain PDSLzg-1, we
designed the appropriate primers. The PCR-amplified gene
was highly homologous to that of the nitrilase of B. pumilus(WP_047202096.1). These genes were highly homologous, andthere were only three amino acid replacements. Then, we tried
to express and purify Bacillus nitrilase. The gene was cloned
into the pET23b vector (pET-BNit). E. coli BL21(DE3) cells
harboring pET-BNit expressed a protein with a molecular massof approximately 30 kDa and could degrade cyanide (Figs. 3aand 3b). Bacillus nitrilase was purified by chromatography.
Interestingly, the purified Bacillus nitrilase appeared as two
different peaks in the size-exclusion chromatograph (Fig. 3c).
Their molecular masses were estimated to be approximately
24 and 509 kDa. Although a large portion of Bacillus nitrilase appeared in the low-molecular-weight fraction, probably
as monomers, it did not exhibit cyanide-degrading activity.
A previous study on Rhodococcus nitrilase has shown thatinactive dimers assemble into a large, functional oligomer,5Biotechnology andApplied Biochemistry

FIG. 5

(a)

(b)

(c)

(d)

Predicted structure of Bacillus nitrilase. (a)Homology-based structure of Bacillus nitrilase.
The catalytic cysteine residue is shown as a spheremodel. (b) Electrostatic surface potential shownwith positive charge colored blue and negative
charge colored red. (c) Catalytic triad of Bacillus
nitrilase. Cys, Glu, and Lys residues that compose
the catalytic triad residues are shown asball-and-stick models. (d) The docking model.
Cyanide is also shown as a ball-and-stick model.

a decamer, in the presence of a substrate benzonitrile [28].
Although we tried to examine the activity of the oligomers,
it was difficult that the oligomer seems to disassemble spontaneously. The relatively low CN degrading activity of E. coli
expressing Bacillus nitrilase compared with B. sp. strain EBE-2
might due to the poor active oligomer formation in E. coli or
existence of other cyanide degrading enzymes in B. sp. strain
EBE-2.
We tried to determine the reaction product of cyanide.
As Bacillus nitrilase did not convert formamide to cyanide(data not shown), it was not a cyanide hydratase. Then, we
examined the formation of formic acid by using formic aciddehydrogenase. As shown in Fig. 3d, conversion of cyanide
to formic acid was observed. Thus, Bacillus nitrilase wasdetermined to be a cyanide hydrolase.
There exist various homologous nitrilases, which areannotated as carbon–nitrogen family hydrolases (Fig. 4).
However, there have been no reports on their cyanide degrading
activity.

6

3.3. Homology modeling
Finally, we tried to make a 3D structural model by homology
modeling. By automated swiss modeling, Bacillus nitrilase
exhibited high sequence identity with the putative carbon–
nitrogen family hydrolase from S. aureus (Fig. 4). Based on its
crystal structure, a homology model was created (Fig. 5a). The
putative catalytic Cys is shown as a spherical model. The thiol ofcysteine attacks the cyano-carbon of nitriles (R–C≡N) to form a
covalent thioimidate complex. The ammonia is released by theaddition of one water molecule. Then the planar thioimidate isconverted to a planar thiol acylenzyme through a tetrahedral
intermediate. The acid product is released by the addition of
a second water molecule and the enzyme is regenerated [29].
The surface charges are shown in Fig. 5b. The positive surface
charge around the catalytic center seemed to be adapted forthe negatively charged cyanide ion. Cys147, Glu41, and Lys112
form the catalytic triad in the catalytic center (Fig. 5c). Glu
acts as the general base for activating the nucleophile of Cys,
and Lys is the catalytic acid responsible for proton transfer tothe substrate. We performed molecular docking simulationsof Bacillus nitrilase and the cyanide ion using the modeled
structure. Among 20 trials, cyanide entered at the putative
catalytic site as one of the most preferred complex structures(Fig. 5d).

Cyanide degrading bacteria and nitrilase

4. Conclusions
We have obtained two cyanide-degrading bacteria from groundwater contaminated with cyanide. Among these bacteria, Bacillus sp. EBE-2 was equipped with a nitrilase gene. Becauseof high cyanide-degrading activity, Bacillus sp. EBE-2 might
be used for the treatment of cyanide contaminated water orsoil. The catalytic site of Bacillus nitrilase contained a typicalcatalytic triad composed of Cys, Glu, and Lys with a positive
surface charge. Similar to other nitrilases, Bacillus nitrilase
also exhibited the activity in the oligomeric configuration. However, the activation mechanism by oligomerization is unknown.
It is possible that some factors, small molecules or proteins, arerequired for the assembly of active oligomers. We found thatthe reaction product was not formamide but formic acid. Thus,
the enzyme was determined to be a cyanide hydrolase.

5. Acknowledgement
This work was partially supported by JSPS KAKENHI Grant
Number 16H04572, 18H04690, and 20H02532. We sincerely
appreciate the technical assistance of Ms. Nina Kurokawa.

6. Accession Numbers
The nucleotide sequences of genes for 16S rRNAs and Bacillus
nitrilase are deposited in the DNA data bank. The accession numbers are LC584259, Bacillus 16S rRNA; LC584260,
Pedobacter 16S rRNA; LC584261, Bacillus nitrilase.

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